KR20230149258A - A combination therapy of GLP-2, and an insulinotropic peptide, TNFα inhibitor, or both for preventing or treating intestinal disease - Google Patents

Abstract

The present invention relates to combination therapy of GLP-2, insulin secreting peptide, TNFα inhibitor, or both for the prevention, improvement or treatment of intestinal diseases.

Description

A combination therapy of GLP-2, and an insulinotropic peptide, TNFα inhibitor, or both for preventing or treating intestinal disease}

The present invention relates to combination therapy of GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both for the prevention or treatment of intestinal diseases.

Intestinal diseases include irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, or intestinal damage. Among these, inflammatory bowel disease (IBD) is an inflammatory disease that causes inflammation or ulcers in the gastrointestinal tract, and causes symptoms such as increased expression of inflammatory cytokines, weight loss, decreased colon length, abdominal pain, fever, diarrhea, and/or bleeding. Symptoms may appear, and these symptoms may repeatedly worsen and improve.

GLP-1, a type of insulin secreting peptide, is an incretin hormone secreted by L-cells in the ileum and colon. The main action of glucagon-like peptide-1 is to increase insulin secretion, and insulin secretion (Glucose dependent secretion) is achieved according to the concentration of glucose, preventing hypoglycemia. Due to these characteristics, it is applied as a treatment method for type 2 diabetes, but because its half-life in the blood is very short, about 2 minutes, it has great potential for development as a drug. Accordingly, a GLP-1 agonist developed and commercially available includes Exendin-4, a GLP-1 analog purified from the salivary glands of glia monster lizards. It has resistance to DPP-IV (Dipeptidyl peptidase-4) and has higher physiological activity than glucagon-like peptide-1, and therefore has a longer half-life in the body than GLP-1, at 2-4 hours (US 5,424,286 A ). However, sufficient duration of physiological activity cannot be expected by simply increasing the resistance of DPP-IV. For example, in the case of exendin-4 (exenatide), which is currently on the market, it is administered to patients by injection twice a day. It must be administered, and the disadvantage of causing vomiting and nausea following administration remains a significant burden to the patient.

GLP-2 is a peptide hormone composed of 33 amino acids produced by L-cells in the small intestine in response to ingested nutrients. GLP-2 induces mucosal growth in the small and large intestines, promotes the growth of enterocytes and crypt cells, and inhibits apoptosis. GLP-2 also increases absorption of nutrients in the small intestine and reduces intestinal permeability. It inhibits gastric emptying and gastric acid secretion, increases intestinal blood flow rate, and relaxes intestinal smooth muscle.

TNFα is a cytokine that plays a central role in the immune response and is also related to inflammatory responses, and abnormal regulation of TNF-α is known to occur in various diseases. Various TNFα inhibitors have been developed to suppress these TNF-α-induced responses.

However, to date, the development of therapeutic agents for intestinal diseases using insulin secreting peptide, GLP-2, and/or TNFα inhibitors is insufficient, and there is a need for continued development of therapeutic agents.

One object of the present invention is to provide a therapy for preventing, improving, or treating intestinal diseases using GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both in combination.

Another object of the present invention is a pharmaceutical composition for the prevention or treatment of intestinal diseases containing GLP-2, wherein the pharmaceutical composition is administered in combination with an insulin secreting peptide, a TNFα inhibitor, or both. To provide a pharmaceutical composition that.

Another object of the present invention is to provide a combination comprising GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both.

Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating intestinal diseases, comprising the above combination.

Another object of the present invention is to provide a pharmaceutical kit for preventing, improving, or treating intestinal diseases, comprising GLP-2, insulin secreting peptide, TNFα inhibitor, or both.

Another object of the present invention is to provide a method for preventing, improving, or treating intestinal diseases, comprising administering and/or using the combination, pharmaceutical composition, or pharmaceutical kit to an individual in need thereof. will be.

Another object of the present invention is to provide a composition containing a pharmaceutically effective amount of GLP-2, a composition containing a pharmaceutically effective amount of an insulinotropic peptide, a composition containing a pharmaceutically effective amount of a TNFα inhibitor, or both. The aim is to provide a method for preventing, improving or treating intestinal diseases, comprising the steps of combined administration and/or combined use to an individual in need.

Another object of the present invention is to use the combination, pharmaceutical composition, or pharmaceutical kit to prevent, improve, or treat intestinal diseases; and/or to provide use for the manufacture of a drug for preventing, improving or treating intestinal diseases.

One aspect embodying the present invention is therapy for preventing, improving, or treating intestinal diseases using GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both in combination.

One embodiment of the present invention is a composition for preventing, improving or treating intestinal diseases containing GLP-2, wherein GLP-2 is administered in combination with an insulin secreting peptide, a TNFα inhibitor, or both. It is a composition made of.

As one embodiment, the present invention is a pharmaceutical composition for preventing, improving or treating intestinal diseases containing a pharmaceutically effective amount of GLP-2, wherein the pharmaceutical composition contains an insulin secreting peptide, a TNFα inhibitor, or both. It is characterized in that it is administered in combination with.

As another embodiment, the present invention is a food composition for preventing or improving intestinal diseases containing GLP-2, wherein the food composition is administered in combination with an insulin secreting peptide, a TNFα inhibitor, or both. do.

In the composition according to any one of the preceding embodiments, the insulin secreting peptide is glucagon-like peptide-1 (GLP-1), exendin-3, exendin-4, and agonists thereof. , derivatives, fragments, variants, and combinations thereof.

In the composition according to any one of the preceding embodiments, the insulin secreting peptide is such that the N-terminal histidine residue of the insulin secreting peptide is imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethyl It is characterized as being an insulin secreting peptide derivative substituted with histidine or β-carboxyimidazopropionyldeshistidine.

In the composition according to any one of the preceding embodiments, the insulin secreting peptide is native exendin-4, an N-terminal amino group bonded to the alpha carbon and alpha carbon of a histidine residue, which is the N-terminal first amino acid of exendin-4. Exendin-4 derivative with the removed, Exendin-4 derivative with the N-terminal amino group of Exendin-4 removed, Exendin-4 derivative with the N-terminal amino group of Exendin-4 replaced with a hydroxyl group, exendin An exendin-4 derivative in which the N-terminal amino group of exendin-4 is modified with two methyl groups, an exendin-4 derivative in which the N-terminal amino group of exendin-4 is replaced with a carboxyl group, the twelfth amino acid of exendin-4 ( It is characterized as an exendin-4 derivative in which lysine is substituted with serine, or an exendin-4 derivative in which the twelfth amino acid (lysine) of exendin-4 is substituted with arginine.

In the composition according to any one of the preceding embodiments, the GLP-2 is characterized in that it is a native GLP-2 or a GLP-2 derivative.

A composition according to any one of the preceding embodiments, wherein the GLP-2 derivative is capable of substituting, adding, deleting, modifying, or modifying at least one amino acid in the native GLP-2 sequence. It is characterized as a GLP-2 derivative in which a modification has occurred selected from the group consisting of a combination of.

In the composition according to any one of the preceding embodiments, the GLP-2 derivative is characterized in that at least one amino acid among amino acids 1, 2, 30, and 33 in SEQ ID NO: 1 is modified.

In the composition according to any one of the preceding embodiments, the GLP-2 derivative is characterized in that it comprises an amino acid sequence represented by the following general formula 1:

[General Formula 1]

_{X 1 _ _}

here,

X ₁ is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine;

X ₂ is alanine, glycine, or Aib(2-aminoisobutyric acid);

X ₃₀ is lysine or arginine;

X ₃₄ is absent or is lysine, arginine, glutamine, histidine, 6-azidolysine or cysteine;

However, among the amino acid sequences of General Formula 1, sequences identical to SEQ ID NO: 1 are excluded.

A composition according to any one of the preceding embodiments, wherein the GLP-2 derivative is

(1) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is cysteine,

(2) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is lysine,

(3) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is arginine, and X ₃₄ is lysine,

(4) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is 6-azidolysine, or

(5) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is arginine, and X ₃₄ is cysteine,

(6) X ₁ is imidazoacetyldeshistidine, X ₂ is Aib, X ₃₀ is lysine, and X ₃₄ is cysteine, or

(7) X ₁ is histidine, X ₂ is Aib, X ₃₀ is lysine, and X ₃₄ is cysteine.

In the composition according to any one of the preceding embodiments, the GLP-2 derivative is characterized in that it comprises an amino acid sequence represented by the following general formula 2:

[General Formula 2]

_{X 1 _ _}

here,

X ₁ is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine;

X ₂ is alanine, glycine, or Aib(2-aminoisobutyric acid);

X ₃₀ is lysine or arginine;

X ₃₄ is one or more optional amino acids or one or more optional amino acids upon which a modification has occurred;

However, among the amino acid sequences of General Formula 2, sequences identical to SEQ ID NO: 1 are excluded.

In the composition according to any one of the preceding embodiments, the GLP-2 derivative is characterized in that it is a peptide with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8.

A composition according to any one of the preceding embodiments, characterized in that GLP-2 and insulin secreting peptide are administered in combination.

The composition according to any one of the preceding embodiments is characterized in that a GLP-2 and a TNFα inhibitor are administered in combination.

A composition according to any one of the preceding embodiments, characterized in that GLP-2, an insulinotropic peptide, and a TNFα inhibitor are administered in combination.

A composition according to any one of the preceding embodiments, wherein the TNFα inhibitor is a soluble TNFa receptor, an anti-TNFα antibody or fragment thereof, or a combination thereof.

The composition according to any one of the preceding embodiments, wherein the intestinal disease is at least one selected from the group consisting of irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, and intestinal damage. It is characterized by

The pharmaceutical composition according to any one of the preceding embodiments, wherein the inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, and Behcet's disease.

A composition according to any one of the preceding embodiments, wherein the composition exhibits one or more of inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration when administered to a subject. do.

A composition according to any one of the preceding embodiments, wherein the composition, when administered to a subject, causes at least one of an increase in the length of the small intestine, a decrease in inflammation in the small intestine, an increase in the length of the large intestine, and a decrease in inflammation in the large intestine.

A composition according to any one of the preceding embodiments, wherein the insulinotropic peptide and GLP-2 are characterized in that their C-terminus is unmodified or amidated.

In the composition according to any one of the preceding embodiments, (i) the insulin secreting peptide is in the form of a long-acting conjugate to which a biocompatible material capable of increasing the in vivo half-life is bound, or

(ii) The GLP-2 is in the form of a long-acting conjugate combined with a biocompatible material that can increase its in vivo half-life, or

(iii) Each of the insulin secreting peptide and GLP-2 is characterized in that it is in the form of a long-acting conjugate to which a biocompatible material capable of increasing the half-life of the insulin secreting peptide and GLP-2 is bound.

In the composition according to any one of the preceding embodiments, the conjugate is characterized by being represented by the following formula (1):

[Formula 1]

X - La - F

here,

X is insulinotropic peptide or GLP-2;

L is a linker containing ethylene glycol repeating units;

a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;

F is the immunoglobulin Fc region;

The “-” above is a covalent bond.

The composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is a non-glycosylated IgG4 Fc region.

A pharmaceutical composition according to any one of the preceding embodiments, wherein F is a dimer consisting of two polypeptide chains, and one end of L is connected to only one polypeptide chain of the two polypeptide chains. .

A composition according to any one of the preceding embodiments, wherein L is polyethylene glycol.

A pharmaceutical composition according to any one of the preceding embodiments, wherein the formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

A composition according to any one of the preceding embodiments, characterized in that the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

A composition according to any one of the preceding embodiments, comprising: (i) GLP-2 and insulin secreting peptide; (ii) GLP-2 and TNFα inhibitors; or (iii) GLP-2, insulinotropic peptide, and TNFα inhibitor are administered in combination simultaneously, sequentially, or in reverse order.

Another embodiment of the present invention is a combination comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both.

Another aspect embodying the present invention is a pharmaceutical composition for preventing, improving, or treating intestinal diseases, comprising the above combination.

Another aspect embodying the present invention is a pharmaceutical kit for preventing, improving, or treating intestinal diseases, including the above combination.

Another aspect embodying the present invention is a method for preventing, improving, or treating enteric disease comprising administering and/or using the combination, pharmaceutical composition, or pharmaceutical kit to an individual in need thereof. .

Another embodiment of the present invention is a composition containing a pharmaceutically effective amount of GLP-2, a composition containing a pharmaceutically effective amount of an insulinotropic peptide, a composition containing a pharmaceutically effective amount of a TNFα inhibitor, or both. It is a method for preventing, improving, or treating intestinal disease, including the steps of administering and/or using both in combination to an individual in need thereof.

Another aspect embodying the present invention is the use of the combination, pharmaceutical composition, or pharmaceutical kit for the prevention, improvement or treatment of enteric diseases, and/or for the manufacture of a medicament for the prevention or treatment of enteric diseases. It is for use.

The combined administration therapy of GLP-2, insulin secreting peptide, TNFα inhibitor, or both according to the present invention has improved effects compared to single administration therapy and can be usefully used for preventing, improving, or treating intestinal diseases.

Figure 1 shows the inhibitory effect on M1 polarization (A) and macrophage differentiation ( This is a diagram confirming the effect of suppressing macrophage differentiation (B) and the effect of suppressing monocyte migration (C).
Figure 2 shows the results of combined administration of GLP-2 (GLP-2 derivative long-acting conjugate), insulinotropic peptide (GLP-1 derivative long-acting conjugate), TNFα inhibitor (anti-TNFα antibody), or both. This diagram shows changes in small intestine length in rats with inflammatory bowel disease caused by indomethacin (INN).
Figure 3 shows indometa following combined administration of GLP-2 (GLP-2 derivative long-acting conjugate), insulinotropic peptide (GLP-1 derivative long-acting conjugate), TNFα inhibitor (anti-TNFα antibody), or both. This diagram shows changes in the inflammation area (ulcer area) in rats with kidney-induced inflammatory bowel disease.
Figure 4 shows mice with ulcerative colitis caused by dextran sulfate sodium (DSS) following combined administration of GLP-2 (GLP-2 derivative long-acting conjugate) and insulinotropic peptide (GLP-1 derivative long-acting conjugate). This diagram shows the change in colon length.
Figure 5 is an ulcerative colitis activity index ( This is a diagram showing the Disease activity index (DAI).

Below, the present invention is described in more detail.

Meanwhile, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Additionally, it cannot be said that the scope of the present invention is limited by the specific description described below.

Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Additionally, such equivalents are intended to be encompassed by this invention.

Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used, as well as generally accepted codes for other amino acids such as Aib (2-aminoisobutyric acid), AZK (6-azidolysine), etc. A three-character code is used. Additionally, amino acids abbreviated herein are described according to the IUPAC-IUB nomenclature.

Alanine Ala, A Arginine Arg, R

Asparagine Asn, N Aspartic acid Asp, D

Cysteine Cys, C Glutamic acid Glu, E

Glutamine Gln, Q Glycine Gly, G

Histidine His, H Isoleucine Ile, I

Leucine Leu, L Lysine Lys, K

Methionine Met, M Phenylalanine Phe, F

Proline Pro, P Serine Ser, S

Threonine Thr, T Tryptophan Trp, W

Tyrosine Tyr, Y Valine Val, V

One embodiment of the present invention is a pharmaceutical composition for preventing, improving or treating intestinal diseases containing GLP-2, wherein the GLP-2 is administered in combination with an insulin secreting peptide, a TNFα inhibitor, or both. Provided is a pharmaceutical composition characterized in that:

In one embodiment of the present invention, the pharmaceutical composition is a pharmaceutical composition for preventing, improving or treating intestinal diseases containing a pharmaceutically effective amount of GLP-2, wherein the pharmaceutical composition contains an insulin secreting peptide (e.g., It may be administered in combination with a pharmaceutically effective amount of an insulin secreting peptide), a TNFα inhibitor (e.g., a pharmaceutically effective amount of a TNFα inhibitor), or both, but is not limited thereto.

Another aspect of the present invention provides the use of GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both in combination to prevent, improve, or treat intestinal diseases.

Another embodiment provides a pharmaceutical composition for preventing, improving, or treating intestinal diseases using GLP-2, an insulin secreting peptide, a TNFα inhibitor, or both in combination.

Specifically, one aspect of the present invention provides a combination, pharmaceutical composition, or pharmaceutical kit containing GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both. In one embodiment, a combination, pharmaceutical composition, or pharmaceutical kit for preventing, improving, or treating intestinal diseases is provided.

In the present invention, the term "combination" refers to the use of combined administration of GLP-2, an insulin secreting peptide, a TNFα inhibitor, or both, and is to be understood as having the same meaning as "combined use." You can. This includes, but is not limited to, pharmaceutical compositions and pharmaceutical kit forms characterized by a combination of GLP-2, insulin secreting peptide, TNFα inhibitor, or both.

Specifically, (i) GLP-2 and insulinotropic peptide; (ii) GLP-2 and TNFα inhibitors; or (iii) GLP-2, insulinotropic peptide, and TNFα inhibitor may be administered in combination simultaneously, sequentially, or in reverse order.

The combined administration is

i) co-administration using a pharmaceutical composition comprising GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both in the form of a mixture or in separate forms,

ii) administering a pharmaceutical composition containing GLP-2, a pharmaceutical composition containing an insulinotropic peptide, a pharmaceutical composition containing a TNFα inhibitor, or both simultaneously, sequentially, or in reverse order, or

iii) A pharmaceutical composition containing two substances among GLP-2, an insulinotropic peptide, and a TNFα inhibitor in the form of a mixture or in separate forms, and the remaining substance is co-administered simultaneously, sequentially, or in reverse order. may, but is not limited to this.

The combination or composition is

a) (i) administered as a mixture of GLP-2 and insulin secreting peptide, (ii) administered as a mixture of GLP-2 and TNFα inhibitor, or (iii) ) administered as a single mixture of GLP-2, insulinotropic peptide, and TNFα inhibitor; or

b) (i) GLP-2 and insulin secreting peptide are administered in separate forms, (ii) GLP-2 and TNFα inhibitor are administered in separate forms, or (iii) GLP-2 and insulin secreting peptides are administered in separate forms. The peptide and TNFα inhibitor are administered in separate forms; or

c) (i) a mixture of GLP-2 and an insulinotropic peptide and a TNFα inhibitor administered in separate forms, or (ii) a mixture of GLP-2 and an insulinotropic peptide and an insulinotropic peptide. may be administered in a separate form, or (iii) a mixture of the insulin secreting peptide and the TNFα inhibitor and GLP-2 may be administered in a separate form, but is not limited thereto.

When in separated form as in b) above, (i) GLP-2 and insulin secreting peptide; (ii) GLP-2 and TNFα inhibitors; or (iii) the GLP-2, insulinotropic peptide, and TNFα inhibitor are each formulated together in separate and separate forms; Alternatively, each may be formulated as a separate preparation and the separate preparations may be administered simultaneously, separately, sequentially, or in reverse order.

In addition, in the case of a separate form as in c) above, the mixture and the insulin secreting peptide, GLP-2, or TNFα inhibitor may be formulated together in separate forms; Alternatively, each may be formulated as a separate preparation and the separate preparations may be administered simultaneously, separately, sequentially, or in reverse order.

In the present invention, “combined administration,” “used in combination,” or “administered in combination” refers not only to simultaneous administration, but also to the administration of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both together in an individual. It should be understood as a dosage form that allows each substance (insulin secreting peptide, GLP-2, and/or TNFα inhibitor) to perform at a level equal to or higher than its original function. Accordingly, when the term “combination” is used herein, it is to be understood that the order is unlimited, indicating simultaneous, separate, sequential, or reverse administration. If the administration is sequential, reverse order, or separate, the order of administration is not particularly limited, but the interval between the second or more component administrations should be such that the beneficial effects of the combination are not lost.

In the present invention, the term "composition comprising a combination" is a combination itself containing the GLP-2 and an insulin secreting peptide, a TNFα inhibitor, or both, or includes the combination and has therapeutic use. It may be, but is not limited to this. For example, it may be used to prevent, improve, or treat intestinal diseases, but is not limited thereto. As used herein, “composition including combination” may be used interchangeably with “composition.”

A composition comprising the combination according to the present invention is for co-administration of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both. It may be formulated as a preparation, or it may be formulated as two or more individual preparations. Specifically, GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both may be administered simultaneously, separately, sequentially, or in reverse order, but are not limited thereto.

In the present invention, the term “kit” may include a combination or composition according to the present invention for combined administration of GLP-2, an insulin secreting peptide, a TNFα inhibitor, or both. Specifically, the kit according to the invention includes a) (i) GLP-2 and insulin secreting peptide, (ii) GLP-2 and TNFα inhibitor, or (iii) GLP-2, insulin secreting peptide, formulated in one agent. , and TNFα inhibitors; b) (i) separate preparations of each of GLP-2 and the insulinotropic peptide, (ii) separate preparations of each of the GLP-2 and the TNFα inhibitor, or (iii) separate preparations of each of the GLP-2, the insulinotropic peptide, and the TNFα inhibitor. ; or c) (i) a formulation of a GLP-2 and an insulinotropic peptide and separate formulations of a TNFα inhibitor, (ii) a formulation of a GLP-2 and a TNFα inhibitor and separate formulations of an insulinotropic peptide, or (iii) ) It may include a preparation containing an insulin secreting peptide and a TNFα inhibitor formulated as one and a separate preparation of GLP-2, and may additionally contain substances required for combined administration of two or more substances, but is not limited thereto.

The present invention provides that when GLP-2, insulin secreting peptide, TNFα inhibitor, or both are used in combination, the effect of preventing, improving, or treating intestinal diseases is significantly greater than that of insulin secreting peptide, GLP-2, and TNFα inhibitor alone. After confirming the increase, the above combination therapy was provided.

The “insulin secreting peptide” refers to a peptide that possesses an insulin secreting function and can stimulate the synthesis or expression of insulin in pancreatic beta cells. The insulin secreting peptide may be, for example, GLP-1 (Glucagon like peptide-1), exendin-3, or exendin-4, but is not limited thereto. The insulin secreting peptide includes not only the native insulin secreting peptide, but also its precursors, agonists, derivatives, fragments, and variants, and their in vivo half-life. It also includes forms of long-acting conjugates combined with biocompatible materials that can increase . The insulin secreting peptide may be included in a pharmaceutical composition in a pharmaceutically effective amount.

GLP-1 is a hormone secreted in the small intestine and generally promotes insulin biosynthesis and secretion, inhibits glucagon secretion, and promotes intracellular glucose absorption. In the small intestine, the glucagon precursor is broken down into three peptides: glucagon, GLP-1, and GLP-2. Here, GLP-1 refers to GLP-1(1-37), which is a form without insulin secretion function and is processed into GLP-1(7-37) form to become active GLP-1(7-37). The amino acid sequence of GLP-1(7-37) is as follows.

GLP-1(7-37):

HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G (SEQ ID NO: 32)

Exendin-3 and Exendin-4 are GLP-1 analogs or derivatives consisting of 39 amino acids showing 53% amino acid sequence similarity to GLP-1, and correspond to insulinotropic peptides. The amino acid sequences of exendin-3 and exendin-4 are as follows.

Exendin-3:

HSDGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO: 33)

Exendin-4:

HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO: 34)

Insulin-secreting peptide derivatives have an insulin-secreting function and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% homology or identity in amino acid sequence with the native insulin-secreting peptide. It may be that some groups of amino acid residues of the insulin secreting peptide have been chemically substituted (e.g. alpha-methylation, alpha-hydroxylation), removed (e.g. deamination) or modified (e.g. N-methylation). It may be in any form, but is not limited thereto.

In one specific embodiment, the insulin secreting peptide derivative of the present invention is synthesized by removing the alpha amino group of the N-terminal histidine, replacing the N-terminal amino group with a hydroxyl group or carboxyl group, N -By removing the N-terminal amino group bound to the alpha carbon and alpha carbon of the terminal histidine, leaving only the imidazo-acetyl functional group, and modifying the N-terminal amino group with two methyl groups, etc. can be manufactured. Insulin secreting peptide derivatives prepared through the above method have the N-terminal histidine residue of the insulin secreting peptide imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β- It may be an insulin secreting peptide derivative substituted with carboxyimidazopropionyldeshistidine.

As one specific example, the insulinotropic peptide derivative may be a GLP-1 derivative, and the GLP-1 derivative includes, but is not limited to, exendin-3, exendin-4, or derivatives thereof.

In addition, an example of an insulinotropic peptide derivative may be a derivative that has chemically mutated the N-terminal amino group or amino acid residue of exendin-4, and the alpha and alpha carbons of the histidine residue, which is the first amino acid at the N-terminus of exendin-4. Imidazoacetyl-deshistidyl-exendin-4 (CA-exendin-4) with the bound N-terminal amino group removed, and desaminohis with the N-terminal amino group of exendin-4 removed. Desaminohistidyl exendin-4 (DA-exendin-4), beta-hydroxyimidazopropionyldeshistidyl exendin-4 (beta-hydroxyimidazopropionyl deshistidyl exendin-4) in which the N-terminal amino group of exendin-4 is replaced with a hydroxyl group β-hydroxyimidazopropionyldeshistidyl exendin-4, HY-exendin-4), N-dimethylhistidyl exendin-4 (DM-exendin) in which the N-terminal amino group of exendin-4 is modified with two methyl groups -4), or beta-carboxyimidazopropionyl-deshistidyl exendin-4 (CX-exendin-4) in which the N-terminal amino group of exendin-4 is substituted with a carboxyl group. , it may be an exendin-4 derivative in which the twelfth amino acid (lysine) of exendin-4 is substituted with serine, or an exendin-4 derivative in which the twelfth amino acid (lysine) of exendin-4 is substituted with arginine. It is not limited to this.

Insulin-secreting peptide fragment refers to a form in which one or more amino acids are removed and/or added to the N-terminus or C-terminus of the native insulin-secreting peptide, and the added amino acids are amino acids that do not exist in nature (e.g., type D). amino acids) are also possible.

Insulin-secreting peptide variants are peptides that differ from the native insulin-secreting peptide in one or more amino acids, and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity. It refers to a peptide that has an insulin secretion function.

An insulin-secreting peptide agonist refers to a substance that binds to the in vivo receptor of the insulin-secreting peptide and exhibits the same biological activity as the insulin-secreting peptide, regardless of the structure of the insulin-secreting peptide.

The production methods used in each of the derivatives, fragments, variants and agents of the present invention can be used independently or in combination. For example, the present invention also includes insulin-secreting peptides that differ in one or more amino acid sequences and have deamination at the N-terminal amino acid residue.

In the present invention, the term "glucagon-like peptide-2" or "GLP-2 (Glucagon-like peptide-2)" is an agonist of the glucagon-like peptide-2 receptor, which is in the form of a polypeptide or can increase its in vivo half-life. It may be in the form of a persistent conjugate in which a biocompatible material is combined, but is not limited thereto.

In the present invention, glucagon-like peptide-2 or GLP-2 includes not only a sequence matching the native human GLP-2, but also a GLP-2 derivative, and a biocompatible material is bound to the native GLP-2 or GLP-2 derivative. It is a concept that also includes continuous forms of conjugation. The GLP-2 may be included in a pharmaceutically effective amount in the composition or in a separate composition so that it can be administered or used in combination with a pharmaceutical composition containing an insulinotropic peptide.

The amino acid sequence of native GLP-2 is as follows.

GLP-2(1-33)

HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 1)

In the present invention, “GLP-2 receptor agonist” refers to a substance that binds to the in vivo or isolated human glucagon-like peptide-2 receptor and produces physiological activity equivalent or similar to that of native GLP-2. For example, GLP-2 agonists may include native GLP-2 or GLP-2 derivatives.

In the present invention, “GLP-2 derivative” refers to a peptide having one or more differences in amino acid sequence compared to native GLP-2; A peptide modified by modifying the natural GLP-2 sequence; and/or mimics of native GLP-2 that have the function of preventing, treating, and/or improving intestinal diseases, such as native GLP-2. Specifically, the GLP-2 derivative is a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof to at least one amino acid in the native GLP-2 sequence. This may have occurred, but is not limited to this.

The added amino acid can be a non-natural amino acid (e.g. D-type amino acid), and non-natural amino acids can be substituted in addition to the natural amino acid. The added amino acid sequence may be derived from native GLP-2, but is not limited thereto. In addition, the modification of amino acids in the present invention includes substitution, addition, removal, or combination of at least one amino acid; or independently, means that some group of amino acid residues has been chemically substituted (e.g. alpha-methylation, alpha-hydroxylation, substitution with an azido group), removed (e.g. deamination) and/or modified (e.g. N-methylation). It can be done, but is not limited to this.

In one specific embodiment, the derivative of GLP-2 of the present invention is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acid sequence to the native GLP-2. It may show homology or identity, and/or some group of amino acid residues of GLP-2 may be chemically substituted (e.g., alpha-methylation, alpha-hydroxylation, substitution with an azido group), removed (e.g., deamination), and/or Alternatively, it may be in a modified form (e.g. N-methylation), but is not limited thereto.

In one specific embodiment, the N-terminal amino group of GLP-2 of the present invention may be substituted, removed, or modified, but is not limited thereto. In order to prevent binding to the N-terminus, which is an important site for the in vivo activity of GLP-2, when preparing a long-acting conjugate of the present invention, a method of removing the alpha amino group of the N-terminal histidine, N-terminal A method of synthesis by substituting an amino group with a hydroxyl or carboxyl group, imidazo-acetyl action by removing the N-terminal amino group bonded to the alpha carbon and alpha carbon of the N-terminal histidine It can be prepared by leaving the base intact, modifying the N-terminal amino group with two methyl groups, etc.

Specifically, the GLP-2 derivative is imidazoacetyl-deshistidyl GLP-2 (imidazoacetyl-deshistidyl-GLP-2) in which the N-terminal amino group bound to the alpha carbon and alpha carbon of the histidine residue, which is the first amino acid at the N-terminal of GLP-2, is removed. GLP-2, CA-GLP-2), desaminohistidyl GLP-2 (DA-GLP-2), with the N-terminal amino group of GLP-2 removed, N-terminal amino group of GLP-2 Beta-hydroxyimidazopropionyldeshistidyl GLP-2 (β-hydroxyimidazopropionyldeshistidyl GLP-2, HY-GLP-2) substituted with a hydroxyl group, the N-terminal amino group of GLP-2 is modified with two methyl groups N-dimethylhistidyl GLP-2 (DM-GLP-2), or beta-carboxyimidazopropionyldeshistidyl-GLP, in which the N-terminal amino group of GLP-2 is replaced with a carboxyl group. It may be -2 (β-carboxyimidazopropionyl-deshistidyl GLP-2, CX-GLP-2), but is not limited thereto. As a non-limiting example, the material structure used to prepare the GLP-2 derivative is as follows.

Herein imidazoacetyldeshistidyl(dine), desaminohistidyl(dine), beta-hydroxyimidazopropionyldeshistidyl(dine), N-dimethylhistidyl(dine), and beta-carboxyimidazo. Propionyldeshistidyl (dine) can be used with the same meaning as imidazoacetyl, des-amino-histidyl, beta-hydroxy-imidazopropionyl, dimethyl-histidyl, and beta-carboxyl-imidazopropionyl, respectively. .

In one specific embodiment, the GLP-2 derivative may be one in which at least one amino acid among amino acids 1, 2, 30, and 33 in SEQ ID NO: 1 has been modified, but is not limited thereto. Specifically, the modification may be a modification selected from the group consisting of substitution, addition, removal, modification, and combinations of at least one amino acid, and in this case, the added amino acid is a non-natural amino acid (e.g., D-type amino acid). is also possible, and substitution of non-natural amino acids in addition to natural amino acids is also possible. The added amino acid sequence may be derived from native GLP-2, but is not limited thereto. In addition, the modification of amino acids in the present invention includes substitution, addition, removal, or combination of at least one amino acid; or independently, means that some group of amino acid residues has been chemically substituted (e.g. alpha-methylation, alpha-hydroxylation, substitution with an azido group), removed (e.g. deamination) and/or modified (e.g. N-methylation). It can be done, but is not limited to this.

In one specific embodiment, the GLP-2 derivative may comprise, but is not limited to, an amino acid sequence of General Formula 1:

[General Formula 1]

_{X 1 _ _}

here,

X ₁ is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine;

X ₂ is alanine, glycine, or Aib(2-aminoisobutyric acid);

X ₃₀ is lysine or arginine;

X ₃₄ is absent or is lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine.

In another specific embodiment, the GLP-2 derivative may comprise, but is not limited to, an amino acid sequence of the following general formula 2:

[General Formula 2]

_{X 1 _ _}

here,

X ₁ is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine;

X ₂ is alanine, glycine, or Aib(2-aminoisobutyric acid);

X ₃₀ is lysine or arginine;

X ₃₄ is one or more arbitrary amino acids or one or more arbitrary amino acids in which a modification has occurred.

Specifically, the amino acid may be a natural amino acid or a non-natural amino acid, and modifications of the amino acid are as described above.

Additionally, among the amino acid sequences of General Formula 1 or 2, sequences identical to SEQ ID NO: 1 may be excluded from GLP-2 derivatives, but are not limited thereto.

Specifically, the GLP-2 derivative of the present invention is the substitution of alanine, the 2nd amino acid of natural GLP-2, with glycine or Aib (2-aminoisobutyric acid), substitution of lysine, the 30th amino acid, with arginine, or these It may have a combination of, but is not limited to this. In addition, the GLP-2 derivative has a thiol group (e.g., cysteine), an amino group (e.g., lysine, arginine, glutamine, or histidine), or an azide group (e.g., 33rd amino acid) at the C-terminus (e.g., amino acid 33). For example, 6-azidolysine) may be introduced, but is not limited thereto.

Since binding occurs at the introduced group when preparing a long-acting conjugate of a GLP-2 derivative, this can be used to prepare a GLP-2 conjugate with a selectively controlled binding site. Specifically, one end of the linker is bound to the hydroxyl, thiol, amino, or azide group of the GLP-2 derivative, and a substance that can increase the in vivo half-life (e.g., immunoglobulin) is attached to the other end of the linker. Fc region) can be bound. The thiol group, amino group, or azide group can be introduced by adding an amino acid to GLP-2, but is not limited thereto. The thiol group is introduced by adding cysteine (C) to GLP-2; Amino groups are introduced by adding lysine (K), arginine (R), glutamine (Q), or histidine (H); The azide group may be introduced by adding 6-azidolysine ( _AZ K), but is not limited thereto.

Specifically, the GLP-2 derivative according to the present invention may have at least one residue of cysteine, lysine, arginine, glutamine, histidine, or 6-azidolysine, but is not limited thereto.

Specifically, the GLP-2 derivative of the present invention includes substitution of alanine, the second amino acid of native GLP-2, with glycine and introduction of a thiol group (e.g., cysteine) at the C-terminus, and more specifically, N - It may include imidazoacetyldeshistidine in which the N-terminal amino group bonded to the alpha carbon and alpha carbon of the histidine residue, which is the first amino acid at the terminal, has been removed, and may have the amino acid sequence of SEQ ID NO: 2, but is not limited thereto. No.

Specifically, the GLP-2 derivative of the present invention includes substitution of alanine, the second amino acid of native GLP-2, with glycine and introduction of an amino group (e.g., lysine) at the C-terminus, and more specifically, N - It may include imidazoacetyldeshistidine in which the N-terminal amino group bonded to the alpha carbon and alpha carbon of the histidine residue, which is the first amino acid at the terminal, has been removed, and may have the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. No.

Specifically, the GLP-2 derivative of the present invention is a substitution of alanine, the 2nd amino acid of native GLP-2, with glycine, substitution of lysine, the 30th amino acid of native GLP-2, with arginine, and a substitution of lysine at the C-terminus. It may include the introduction of an amino group (e.g., lysine), and more specifically, it may include imidazoacetyldeshistidine in which the N-terminal amino group bonded to the alpha carbon and alpha carbon of the histidine residue, which is the first N-terminal amino acid, has been removed. For example, it may have the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

Specifically, the GLP-2 derivative of the present invention includes substitution of alanine, the second amino acid of native GLP-2, with glycine and introduction of an azide group (e.g., 6-azidolysine) at the C-terminus. , More specifically, it may include imidazoacetyldeshistidine in which the N-terminal amino group bound to the alpha carbon and alpha carbon of the histidine residue, which is the first amino acid at the N-terminus, has been removed, and as an example, it may have the amino acid sequence of SEQ ID NO: 5. However, it is not limited to this.

Specifically, the GLP-2 derivative of the present invention is a substitution of alanine, the 2nd amino acid of native GLP-2, with glycine, substitution of lysine, the 30th amino acid of native GLP-2, with arginine, and a substitution of lysine at the C-terminus. It may include the introduction of a thiol group (e.g., cysteine), and more specifically, it may include imidazoacetyldeshistidine in which the N-terminal amino group bonded to the alpha carbon and alpha carbon of the histidine residue, which is the N-terminal first amino acid, has been removed. For example, it may have the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

Specifically, the GLP-2 derivative of the present invention includes substitution of alanine, the second amino acid of native GLP-2, with 2-aminoisobutyric acid and introduction of a thiol group (e.g., cysteine) at the C-terminus, For example, it may have the amino acid sequence of SEQ ID NO: 8, and more specifically, it may include imidazoacetyldeshistidine in which the N-terminal amino group bound to the alpha carbon and alpha carbon of the histidine residue, which is the first N-terminal amino acid, has been removed, , for example, may have the amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

The GLP-2 derivatives of SEQ ID NOs: 2 to 8 are shown in Table 1 below.

In Table 1, _ca H is substituted with imidazoacetyldeshistidine instead of histidine, Aib is 2-aminoisobutyric acid, and _AZ K is 6-azido-L-lysine (6-azido- refers to L-lysyine).

GLP-2 according to the present invention may be a peptide containing the specific sequence described above, or a peptide composed of (or essentially composed of) the specific sequence described above, but is not limited thereto.

Meanwhile, even if it is described herein as a peptide or GLP-2 ‘consisting of a specific sequence number’, if it has the same or corresponding activity as a peptide or GLP-2 consisting of the amino acid sequence of the sequence number, the corresponding It does not exclude meaningless sequence additions before or after the amino acid sequence of the sequence number, mutations that may occur naturally, or silent mutations thereof, and it is obvious that even if such sequence additions or mutations are present, they are within the scope of the present application. .

Specifically, the GLP-2 derivative has the general formula 1 or 2: (1) X ₂ is glycine _or Aib, (2) X ₃₀ is lysine or arginine, or (3) ₃₀ may be lysine or arginine, but is not limited thereto.

Specifically, GLP-2 derivatives have general formula 1 or 2

(1) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is cysteine,

(2) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is lysine,

(3) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is arginine, and X ₃₄ is lysine,

(4) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is 6-azidolysine, or

(5) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is arginine, and X ₃₄ is cysteine,

(6) X ₁ is imidazoacetyldeshistidine, X ₂ is Aib, X ₃₀ is lysine, and X ₃₄ is cysteine, or

(7) X ₁ may be histidine, X ₂ may be Aib, X ₃₀ may be lysine, and X ₃₄ may be cysteine, but is not limited thereto.

In the present invention, such modifications for producing agonists, fragments, variants and derivatives of native GLP-2 include modification using L-type or D-type amino acids, and/or non-natural amino acids; and/or modification of the native sequence by modification or post-translational modification (e.g., methylation, acylation, ubiquitination, intramolecular covalent bonding, etc.).

In the present invention, the N-terminus and/or C-terminus of the GLP-2 or GLP-2 derivative is chemically modified or protected with an organic group in order to protect against protein-cleaving enzymes in vivo and increase stability. Alternatively, GLP-2 or a GLP-2 derivative may be in a modified form with amino acids added to the terminal, etc.

In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, so to remove these charges, the N-terminus is acetylated and/or the C-terminus is amidated. It can be done, but is not particularly limited thereto. Specifically, in the present invention, GLP-2 or a GLP-2 derivative may have its C-terminus unmodified or amidated, but is not limited thereto.

In the present invention, the term "TNFα (Tumor Necrosis Factor alpha) inhibitor" refers to substances that reduce the activity of TNFα, and is used in combination therapy with GLP-2 or with GLP-2 and insulin secreting peptide to increase the intestinal activity compared to alone. It includes without limitation substances that can prevent, improve or treat diseases. Specifically, the TNFα inhibitor is a substance that reduces the activity of TNFα by binding to TNFα or binding to the receptor of TNFα and ultimately interfering with the binding between TNFα and its receptor; Alternatively, it may be a substance that reduces the activity of TNFα by reducing the production of TNFα in cells, and this substance is not limited to its form, such as a compound, nucleic acid, or peptide.

As one specific example, TNFα inhibitors include soluble TNF receptors (e.g., etanercept), anti-TNFα antibodies or fragments thereof (e.g., infliximab, adalimumab, certolizumab) pegol (certolizumab pegol, golimumab)], compounds (e.g., thalidomide and its derivatives (e.g., lenalidomide, pomalidomide)); xanthine and its derivatives (eg, pentoxifylline), bupropion; 5-HT _2A agonists (e.g., (R)-DOI ((R)-2,5-Dimethoxy-4-iodoamphetamine), TCB-2, Lysergic acid diethylamide (LSD), LA-SS-Az (Lysergic acid 2,4-dimethylazetidide))], or a combination thereof, but is not limited thereto. In addition to these substances, substances known in the art to reduce the activity of TNFα can also be used as TNFα inhibitors herein. Specifically, the TNFα inhibitor may be a soluble TNF receptor, an anti-TNFα antibody or fragment thereof, or a combination thereof, but is not limited thereto.

In the present invention, the insulin secreting peptide and/or GLP-2 may be in the form of a long-acting conjugate combined with a biocompatible material capable of increasing the half-life in vivo, but is not limited thereto. The long-acting conjugate may exhibit increased persistence of efficacy compared to a derivative of insulin secreting peptide or GLP-2 to which a biocompatible material (e.g., immunoglobulin Fc region) is not bound, and in the present invention, insulin secreting peptide or GLP-2 A conjugate containing an insulin-secreting peptide or GLP-2 whose half-life is increased by binding a biocompatible material to -2 is referred to as “insulin-secreting peptide long-acting conjugate or long-acting insulin-secreting peptide conjugate” or “GLP-2 long-acting conjugate”, respectively. It is referred to as “conjugate or long-acting conjugate of GLP-2.” In the present invention, persistent conjugate is used interchangeably with conjugate.

In one embodiment,

(i) the insulin secreting peptide is in the form of a long-acting conjugate in which a biocompatible material capable of increasing its in vivo half-life is bound, or

(ii) The GLP-2 is in the form of a long-acting conjugate combined with a biocompatible material that can increase its in vivo half-life, or

(iii) Each of the insulin secreting peptide and GLP-2 may be in the form of a long-acting conjugate in which a biocompatible material that can increase the half-life of the insulin secreting peptide and GLP-2 is combined.

Meanwhile, this conjugate may be non-naturally occurring.

Additionally, in the long-acting conjugate, the connection between the insulin secreting peptide or GLP-2 and the biocompatible material (e.g., immunoglobulin Fc region) may be a physical or chemical bond, a non-covalent bond, or a covalent bond, and may specifically be a covalent bond. However, it is not limited to this.

In addition, the method of connecting the insulin secreting peptide or GLP-2 and the biocompatible material (e.g., immunoglobulin Fc region) in the long-acting conjugate is not particularly limited, but the insulin secreting peptide or GLP-2 and the biocompatible material (e.g., immunoglobulin Fc region) are linked through a linker. For example, immunoglobulin Fc regions) may be linked together.

Additionally, Korean Patent Publication No. 10-2019-0037181 regarding long-acting conjugate of GLP-2 is incorporated herein by reference.

In one specific embodiment, the insulinotropic peptide or long-acting conjugate of GLP-2 of the present invention may have the structure of Formula 1 below, but is not limited thereto.

[Formula 1]

X - La - F

here,

X is insulinotropic peptide or GLP-2;

L is a linker (e.g., a linker containing ethylene glycol repeat units);

a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;

F is a biocompatible material (e.g., immunoglobulin Fc region) that can increase the in vivo half-life of X;

The “-” is a chemical bond (eg, a covalent bond).

More specifically, .

Additionally, F may be directly connected to

In the conjugate according to the present invention, F is a substance capable of increasing the half-life of

The F may be bonded to X through a covalent chemical bond or a non-covalent chemical bond, and F and X may be bonded to each other through L through a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

The substances that can increase the in vivo half-life of , antibody fragments, FcRn binding substances, in vivo connective tissues, nucleotides, fibronectin, transferrin, saccharide, heparin, and elastin, but are not particularly limited thereto.

In the case of the elastin, it may be human tropoelastin, which is a water-soluble precursor, and may be a polymer of some sequences or some repeating units, including, but not limited to, elastin-like polypeptides. .

Examples of the above polymers include polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, Examples include high molecular weight polymers selected from the group consisting of hyaluronic acid, oligonucleotides, and combinations thereof, and the polysaccharide may include dextran, but is not particularly limited thereto.

As used herein, polyethylene glycol is a term encompassing all forms of ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.

Additionally, the biocompatible material includes poly-amino acids such as poly-lysine, poly-aspartic acid, and poly-glutamic acid, but is not limited thereto.

Additionally, the fatty acid may have binding ability to albumin in vivo, but is not particularly limited thereto.

As an example of the F, the F may be an FcRn binding substance, specifically, the FcRn binding substance may be an immunoglobulin Fc region, more specifically an IgG Fc region, more specifically a non-glycosylated IgG4 Fc It may be a region, but is not particularly limited thereto.

As a specific example of the present invention, the F (e.g., immunoglobulin Fc region) is a dimer composed of two polypeptide chains, and one end of L is connected to only one polypeptide chain of the two polypeptide chains. It may have, but is not limited to this.

In one specific embodiment, the long-acting conjugate of the present invention may be one in which an insulin secreting peptide or GLP-2 and an immunoglobulin Fc region are linked, but is not limited thereto.

In the present invention, “immunoglobulin Fc region” refers to the heavy chain constant region excluding the heavy and light chain variable regions of immunoglobulin. Specifically, the immunoglobulin Fc region may include a portion of heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3), and more specifically, a hinge region (meaning the entire or part of the hinge region) It may further include .). The immunoglobulin Fc region may be a component of the moiety of the conjugate of the present invention. Specifically, it corresponds to F in Formula 1 above.

In this specification, the Fc region refers to not only the native sequence obtained from papain digestion of immunoglobulin, but also its derivatives and substitutions, such as one or more amino acid residues in the native sequence converted by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. This includes variants, such as sequences that are different from the natural type. The above derivatives, substitutions, and variants are assumed to have the ability to bind to FcRn.

The F (e.g., immunoglobulin Fc region) has a structure in which two polypeptide chains are connected by a disulfide bond, and may have a structure in which they are connected only through the nitrogen atom of one of the two chains, but is limited to this. It doesn't work. The connection through the nitrogen atom may be connected to the epsilon amino atom or N-terminal amino group of lysine through reductive amination.

A reductive amination reaction refers to a reaction in which the amine group or amino group of a reactant reacts with the aldehyde (i.e., a functional group capable of reductive amination) of another reactant to produce an amine, and then forms an amine bond through a reduction reaction. It is an organic synthesis reaction widely known in the technical field.

As one specific example, the immunoglobulin Fc region may be connected through its N-terminal nitrogen atom.

This immunoglobulin Fc region may include a hinge portion in the heavy chain constant region, but is not limited thereto.

In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.

As used herein, the term “hinge sequence” refers to a region located in the heavy chain that forms a dimer of the immunoglobulin Fc region through an inter disulfide bond.

In the present invention, the hinge sequence may be mutated to have only one cysteine residue by deleting part of the hinge sequence having the following amino acid sequence, but is not limited thereto:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 11).

The hinge sequence may include only one cysteine residue due to the deletion of the 8th or 11th cysteine residue in the hinge sequence of SEQ ID NO: 11. The hinge sequence of the present invention may be composed of 3 to 12 amino acids, including only one cysteine residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 12), Glu-Ser- Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 13), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 14), Glu- Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 15), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 16), Glu-Ser-Lys- Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 17), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 18), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 19) , Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 20), Pro-Ser-Cys-Pro (SEQ ID NO: 21), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: Number 22), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 23), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 24), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 25), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 26), Glu-Ser-Lys-Pro-Ser- Cys-Pro (SEQ ID NO: 27), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 28), Glu-Pro-Ser-Cys (SEQ ID NO: 29), Ser-Cys-Pro (SEQ ID NO: 30).

More specifically, the hinge sequence may include the amino acid sequence of SEQ ID NO: 21 (Pro-Ser-Cys-Pro) or SEQ ID NO: 30 (Ser-Cys-Pro), but is not limited thereto.

In a more specific form of the long-acting conjugate of the present invention, the N-terminus of the immunoglobulin Fc region in the conjugate is proline, and in this conjugate, the Fc region is connected to a linker through the nitrogen atom of the proline.

In one specific embodiment, the immunoglobulin Fc region may be in the form of a dimer in which two chains of the immunoglobulin Fc region form a homodimer or heterodimer due to the presence of a hinge sequence. The conjugate of Formula 1 of the present invention may be in a form where one end of the linker is connected to one chain of the immunoglobulin Fc region of the dimer, but is not limited thereto.

As used herein, the term “N-terminus” refers to the amino terminus of a protein or polypeptide, and is the most terminal of the amino terminus, or 1, 2, 3, 4, 5, 6, from the most terminal. It may contain up to 7, 8, 9, or 10 or more amino acids. The immunoglobulin Fc region of the present invention may include, but is not limited to, a hinge sequence at the N-terminus.

In addition, the immunoglobulin Fc region of the present invention, as long as it has substantially the same or improved effect as the natural type, excludes only the heavy and light chain variable regions of immunoglobulin and includes part or all of the heavy chain constant region 1 (CH1) and/or light chain constant region. 1 (CL1). Additionally, it may be a region in which some fairly long amino acid sequences corresponding to CH2 and/or CH3 have been removed.

For example, the immunoglobulin Fc region of the present invention includes 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) It can be a combination of one or two or more of the CH1 domain, CH2 domain, CH3 domain, and CH4 domain with the immunoglobulin hinge region (or part of the hinge region), or 6) a dimer of each domain of the heavy chain constant region and the light chain constant region. However, it is not limited to this.

In addition, as one embodiment of the persistent conjugate of the present invention, the immunoglobulin Fc region may be in a dimeric form, and one molecule of X may be covalently linked to one Fc region in the dimeric form, , In this case, the immunoglobulin Fc and X may be linked to each other by one and the same linker. Meanwhile, it is also possible for two X molecules to bind symmetrically to one Fc region in the form of a dimer. At this time, the immunoglobulin Fc and X may be connected to each other by a linker. However, it is not limited to the examples described above.

Additionally, the immunoglobulin Fc region of the present invention includes not only the native amino acid sequence but also sequence derivatives thereof. Amino acid sequence derivative means that one or more amino acid residues in the natural amino acid sequence have a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322 or 327 to 331, which are known to be important for binding, can be used as suitable sites for modification.

In addition, various types of derivatives are possible, such as in which the site capable of forming a disulfide bond is removed, several amino acids at the N-terminus of native Fc are removed, or a methionine residue is added to the N-terminus of native Fc. do. Additionally, to eliminate the effector function, the complement binding site, for example, the C1q binding site, may be removed, and the ADCC (antibody dependent cell mediated cytotoxicity) site may be removed. Techniques for producing sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication No. WO 97/34631, International Patent Publication No. 96/32478, etc.

Amino acid exchanges in proteins and peptides that do not overall alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. In some cases, it is modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation ( modification) may occur.

The above-described Fc derivative may exhibit biological activity equivalent to that of the Fc region of the present invention and may have increased structural stability of the Fc region against heat, pH, etc.

In addition, this Fc region may be obtained from a natural type isolated in vivo from animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or may be obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof. Here, the method of obtaining from the natural type may be a method of obtaining the entire immunoglobulin by isolating it from a human or animal body and then treating it with a proteolytic enzyme. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

Additionally, the immunoglobulin Fc region may be in the form of native sugar chains, increased sugar chains compared to the native type, decreased sugar chains compared to the native type, or sugar chains removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase, decrease, or remove these immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc region with the sugar chain removed from the Fc has a significantly reduced binding ability to complement (c1q) and reduces or eliminates antibody-dependent cytotoxicity or complement-dependent cytotoxicity, so it does not induce unnecessary immune responses in vivo. No. In this respect, a form that is more suitable for its original purpose as a drug carrier would be an immunoglobulin Fc region in which sugar chains have been removed or non-glycosylated.

In the present invention, "Deglycosylation" refers to an Fc region in which sugars have been removed with an enzyme, and aglycosylation refers to an Fc region produced in prokaryotes, or in a more specific embodiment, Escherichia coli, and not glycosylated. .

Meanwhile, the immunoglobulin Fc region may be of human or animal origin such as cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment, may be of human origin.

Additionally, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to improve the half-life of ligand binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

In addition, as one specific example, the immunoglobulin Fc region is a fragment of human IgG4 Fc, and is a homodimer in which two monomers are linked through a disulfide bond (inter-chain form) between cysteine, the 3rd amino acid of each monomer. In this case, the homodimer may have/have a disulfide bond between cysteines 35 and 95 and between cysteines 141 and 199 in each monomer, that is, two disulfide bonds (intra-chain form).

The number of amino acids in each monomer may consist of 221 amino acids, and the amino acids forming a homodimer may consist of a total of 442 amino acids, but is not limited thereto. Specifically, the immunoglobulin Fc region is a homodimer containing the amino acid sequence of SEQ ID NO: 35 (consisting of 442 amino acids) (here, two monomers having the amino acid sequence of SEQ ID NO: 31 (consisting of 221 amino acids) are 3 of each monomer. A homodimer is formed through a disulfide bond between the amino acids cysteine, and the monomers of the homodimer independently form an internal disulfide bond between the cysteines at positions 35 and 95, and an internal disulfide bond between the cysteines at positions 141 and 199. ), but is not limited thereto.

Meanwhile, in the present invention, “combination” related to the immunoglobulin Fc region means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of a different origin. It means to do. That is, it is possible to prepare a dimer or multimer from two or more Fc regions selected from the group consisting of Fc regions of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE.

In the present invention, “hybrid” is a term meaning that sequences corresponding to immunoglobulin Fc regions of two or more different origins exist within the single-chain immunoglobulin constant region. In the case of the present invention, various types of hybrids are possible. That is, a domain hybrid consisting of 1 to 4 domains from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc is possible, and may include a hinge.

Meanwhile, IgG can also be divided into subclasses of IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations of these or hybridization thereof are also possible. Specifically, the IgG2 and IgG4 subclasses, and most specifically, the Fc region of IgG4, which has almost no effector function such as complement dependent cytotoxicity (CDC).

In addition, the persistence of the effect of the above-described conjugate may be increased compared to native insulin secreting peptide or native GLP-2, or compared to This includes, but is not limited to, forms encapsulated in nanoparticles.

In Formula 1, linker L may be a peptide linker or a non-peptide linker (for example, a linker containing an ethylene glycol repeating unit).

When L is a peptidic linker, it may contain one or more amino acids, for example, from 1 to 1000 amino acids, but is not particularly limited thereto. In the present invention, various known peptide linkers can be used to connect F and It may be a natural number greater than or equal to 1. However, it is not limited to the above example.

In the present invention, “non-peptide linker” includes a biocompatible polymer in which two or more repeating units are linked. The repeating units are linked to each other through arbitrary covalent bonds rather than peptide bonds. The non-peptide linker may be a component of the moiety of the conjugate of the present invention and corresponds to L in Formula 1 above.

The non-peptide linker that can be used in the present invention can be used without limitation as long as it is a polymer that is resistant to proteolytic enzymes in vivo.

In the present invention, the non-peptide linker can be used interchangeably with a non-peptide polymer.

In addition, without being particularly limited thereto, the non-peptide linker may be polyethylene glycol, polypropylene glycol, copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide (e.g., dextran, etc.), poly It may be selected from the group consisting of biodegradable polymers such as vinyl ethyl ether, polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

In addition, the non-peptide linker that can be used in the present invention can be used without limitation as long as it is a polymer that is resistant to proteolytic enzymes in vivo. Specifically, the molecular weight of the non-peptide linker is in the range of more than 0 to about 100 kDa, and about 1 to about 100. kDa range, specifically, but not limited to, about 1 to 50 kDa, about 1 to about 20 kDa, about 1 to about 10 kDa, or about 3.4 kDa to 10 kDa.

Although not specifically limited thereto, the non-peptidyl linker may be a linker containing an ethylene glycol repeating unit, for example, polyethylene glycol, and may also be a derivative thereof already known in the art and easily available at the level of skill in the art. Derivatives that can be prepared are also included within the scope of the present invention.

The repeating unit of the non-peptide linker may be an ethylene glycol repeating unit. Specifically, the non-peptide linker may include an ethylene glycol repeating unit and a functional group used in the production of a conjugate at the terminal. The persistent conjugate according to the present invention may be in the form where X and F are connected through the above functional group, but is not limited thereto. In the present invention, the non-peptide linker may include two, three or more functional groups, and each functional group may be the same or different from each other, but is not limited thereto.

Specifically, the linker may include a repeating unit represented by the following formula (4). An example may be, but is not limited to, polyethylene glycol (PEG):

[Formula 4]

Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited thereto.

In the persistent conjugate, the PEG moiety may include not only the -(CH ₂ CH ₂ O) _n - structure but also an oxygen atom intervening between the linking element and the -(CH ₂ CH ₂ O) _n - structure. It is not limited.

Additionally, in one specific embodiment, the conjugate may be a structure in which an insulin secreting peptide or GLP-2 and an immunoglobulin Fc region (F) are covalently linked through a linker containing an ethylene glycol repeat unit, but is limited thereto. no.

The polyethylene glycol is a term encompassing all forms of ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.

In one embodiment, the ethylene glycol repeating unit may be expressed as [OCH ₂ CH ₂ ]n, where the value of n is a natural number and is the average molecular weight of the [OCH ₂ CH ₂ ]n site in the peptide conjugate, such as a number. The average molecular weight may be set to be greater than O to about 100 kDa, but is not limited thereto. As another example, the n value is a natural number and the average molecular weight of the [OCH ₂ CH ₂ ]n site in the peptide conjugate, for example, the number average molecular weight is about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa , about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, about 3 to about 15 kDa, about 3 to about 13 kDa, about 3 to about 11 kDa, about 3 to about 10 kDa, About 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.4 kDa, about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa, about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9.5 to about 10.5 kDa, about 3.4 kDa, or about 10 kDa. , but is not limited to this.

In the present invention, the term "about" is a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range that are equivalent or similar to the value following the term "about." Not limited.

In addition, the non-peptide linker of the present invention that binds to the immunoglobulin Fc region may be a combination of not only one type of polymer but also different types of polymers.

In one specific embodiment, both ends of the non-peptide linker may bind to the thiol group, amino group, and hydroxyl group of the immunoglobulin Fc region and the thiol group, amino group, azide group, and hydroxyl group of GLP-2. , but is not limited to this.

Specifically, the non-peptide linker has a reactive group capable of binding to immunoglobulin Fc and insulin secreting peptide or GLP-2 at both ends, specifically, a thiol group of cysteine in the immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine and/or histidine; And/or bonded to the hydroxyl group located at the C-terminus, and the thiol group of the cysteine of GLP-2; amino groups of lysine, arginine, glutamine and/or histidine; Azide group of azidolysine; and/or a reactive group that can be bonded to a hydroxyl group, but is not limited thereto.

More specifically, the reactive group of the non-peptide polymer may be one or more selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

In the above, examples of the aldehyde group include propionic aldehyde group or butyl aldehyde group, but are not limited thereto.

In the above, succinimide derivatives include succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N -Hydroxysuccinimide, hydroxysuccinimidyl or succinimidyl carbonate may be used, but is not limited thereto.

The non-peptide linker can be converted into a non-peptide polymer linkage by connecting a biocompatible material (eg, immunoglobulin Fc) and an insulin secreting peptide or GLP-2 through the above-described reactive group.

Additionally, the final product resulting from reductive alkylation via an aldehyde linkage is much more stable than that linked via an amide linkage. The aldehyde reactive group reacts selectively at the N-terminus at low pH, and can form a covalent bond with a lysine residue at high pH conditions, for example, pH 9.0.

The terminal reactive groups of the non-peptide linker of the present invention may be the same or different from each other. The non-peptide linker may have an aldehyde group reactive group at the terminal, and the non-peptide linker may have an aldehyde group and a maleimide reactive group at the terminal, respectively, or may have an aldehyde group and a succinimide reactive group at the terminal, respectively. may, but is not limited to this.

For example, it may have a maleimide group at one end and an aldehyde group, propionic aldehyde group, or butyl aldehyde group at the other end. As another example, it may have a succinimidyl group at one end and a propionic aldehyde group or butyl aldehyde group at the other end.

When polyethylene glycol having a hydroxy reactive group at the propion end is used as a non-peptidyl linker, the hydroxy group can be activated into the various reactive groups through a known chemical reaction, or polyethylene glycol with a commercially available modified reactive group can be used as a non-peptide linker. The conjugate of the present invention can be manufactured using.

In one specific embodiment, the reactive group of the non-peptidyl linker may be connected to a cysteine residue of the insulin secreting peptide or GLP-2, more specifically, the -SH group of cysteine, but is not limited thereto.

If maleimide-PEG-aldehyde is used, the maleimide group is connected to the -SH group of the insulin secreting peptide or GLP-2 through a thioether bond, and the aldehyde group is reductively alkylated with the -NH ₂ group of immunoglobulin Fc. It can be connected through a reaction, but it is not limited to this, and this corresponds to one example.

Through this reductive alkylation, the N-terminal amino group of the immunoglobulin Fc region is connected to the oxygen atom located at one end of PEG through a linker functional group having the structure of -CH ₂ CH ₂ CH ₂ -, -PEG-O -CH ₂ CH ₂ CH ₂ NH-Can form a structure similar to immunoglobulin Fc, and can form a structure where one end of PEG is linked to GLP-2 or a sulfur atom located on the cysteine of GLP-2 through a thioether bond. You can. The above-mentioned thioether bond is It may include the structure of

However, there is no particular limitation to the above-described example, and this corresponds to one example.

Additionally, in the above complex, the reactive group of the non-peptide linker may be connected to -NH ₂ located at the N-terminus of the immunoglobulin Fc region, but this corresponds to one example.

Additionally, in the above conjugate, the insulin secreting peptide or GLP-2 may be linked to a non-peptide linker having a reactive group through the C-terminus, but this is one example.

In the present invention, “C-terminus” refers to the carboxy terminus of a peptide, and for the purposes of the present invention, it refers to a position that can bind to a non-peptide polymer. For example, but not limited thereto, it may include not only the most terminal amino acid residue of the C-terminus, but also all amino acid residues around the C-terminus, and may specifically include the first to 20th amino acid residues from the most terminal. You can.

As one specific example, the conjugate of Formula 1 may have the structure of Formula 2 or 3 below.

[Formula 2]

[Formula 3]

In Formula 2 or 3, X is the peptide of Formula 1 described above;

F is the immunoglobulin Fc fragment;

n may be a natural number. At this time, the description of n is the same as described above.

In one specific example, the long-acting conjugate of Formula 2 is a structure in which In the succinimide ring, F may be connected to the oxypropylene group of Formula 2. In addition, the persistent conjugate of Formula 3 is a structure in which X of the insulin secreting peptide or GLP-2 and F of the immunoglobulin Fc region are covalently linked through an ethylene glycol repeat, where , F may be connected to another oxypropylene group in Formula 3.

In Formula 2 or 3, the value of n is determined such that the average molecular weight of the [OCH ₂ CH ₂ ]n site in the peptide conjugate, for example, the number average molecular weight is 1 to 100 kDa, or 1 to 20 kDa or 10 kDa. It may be, but is not limited to this.

In one embodiment, the site where X is connected to the succinimide ring of Formula 2 or the succinimide ring of Formula 2 may be a sulfur atom of the C-terminal cysteine of Additionally, the oxypropylene group of Formula 3 or the site where X is connected to the oxypropylene group of Formula 3 may be the sulfur atom of the C-terminal cysteine of X.

The portion within F that is connected to the oxypropylene group of Formula 2 or Formula 3 is not particularly limited. In one embodiment of the present invention, the portion of F connected to the oxypropylene group may be the N-terminal nitrogen or the nitrogen atom of an internal residue of F (e.g., the epsilon nitrogen of lysine). In one specific embodiment of the present invention, the site where F is connected to the oxypropylene group of Formula 2 or Formula 3 may be the N-terminal proline of F, but is not limited thereto.

Meanwhile, the insulin secreting peptide, GLP-2, long-acting conjugate thereof, or TNFα inhibitor according to the present invention may be in the form of itself, a salt thereof (e.g., a pharmaceutically acceptable salt), or a solvate thereof. Includes.

Additionally, the insulinotropic peptide, GLP-2, long-acting conjugate thereof, or TNFα inhibitor may be in any pharmaceutically acceptable form.

The type of salt is not particularly limited. However, it is desirable to be in a form that is safe and effective for individuals, such as mammals, but is not particularly limited thereto.

In the present invention, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, and formic acid. , benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

Additionally, the term “solvate” used in the present invention refers to a complex in which the peptide, compound, or salt thereof according to the present invention forms a complex with a solvent molecule.

The natural insulin secreting peptide, modified insulin secreting peptide, GLP-2 and GLP-2 derivatives, and TNFα inhibitor of the present invention can be synthesized through solid phase synthesis, can also be produced by recombinant methods, and are manufactured by commercial request. You can do it, and you can use what is sold commercially.

As another embodiment, the combination, pharmaceutical composition, or pharmaceutical kit of the present invention

(i) comprises GLP-2 and insulinotropic peptide, or

Contains GLP-2 and TNFα inhibitors,

Contains GLP-2, insulinotropic peptide, and TNFα inhibitors;

(ii) GLP-2; and an insulin secreting peptide long-acting conjugate to which a biological substance capable of increasing the in vivo half-life is bound, or

Contains GLP-2, the insulin secreting peptide long-acting conjugate, and a TNFα inhibitor, or

(iii) GLP-2 long-acting conjugate combined with a biocompatible material that can increase the in vivo half-life; and an insulinotropic peptide,

Contains the GLP-2 long-acting conjugate and TNFα inhibitor,

Containing the GLP-2 long-acting conjugate, insulin secreting peptide, and TNFα inhibitor,

(iv) GLP-2 long-acting conjugate combined with a biocompatible material that can increase the in vivo half-life; and an insulin secreting peptide long-acting conjugate combined with a biocompatible material capable of increasing the in vivo half-life, or

It may include the GLP-2 long-acting conjugate, the insulin secreting peptide long-acting conjugate, and a TNFα inhibitor.

The combination, pharmaceutical composition, or pharmaceutical kit of the present invention can be used to prevent, improve, or treat intestinal diseases.

As used herein, the term “prevention” refers to a combination comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both; Or, “treatment” refers to any act of suppressing or delaying the onset of a desired disease, such as an intestinal disease, by administering a composition combining GLP-2 with an insulin secreting peptide, a TNFα inhibitor, or both, and “treatment” refers to the combination of the above. Alternatively, it refers to any action in which the symptoms of a desired disease, such as an intestinal disease, are improved or benefited by administration of the composition. Additionally, “improvement” refers to any action that results in at least a decrease in the severity of a parameter, such as a symptom, associated with the condition being treated by administration of the combination or composition of the present invention.

In the present invention, the term "administration" refers to introducing a predetermined substance into a patient by any appropriate method, and the route of administration of the composition is not particularly limited, but the GLP-2, insulin secreting peptide, and TNFα inhibitor are used in the body. It can be administered through any common route that can reach the target, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration. It can be administered, rectally administered, etc.

In the method of the present invention, the GLP-2, insulin secreting peptide, and TNFα inhibitor may be administered through the same administration route or may be administered through different administration routes, and the administration routes of the drugs administered in combination may be independent of each other. .

The GLP-2, insulin secreting peptide, TNFα inhibitor, or both can be used in combination to prevent or treat intestinal diseases.

The intestinal disease may be at least one selected from the group consisting of irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, and intestinal damage, but can be prevented, improved, or prevented by the composition of the present invention. Enteric diseases treated include without limitation.

As a specific example, the intestinal disease may be inflammatory bowel disease. The inflammatory bowel disease (IBD) is an inflammatory disease in which inflammation or ulcers occur in the gastrointestinal tract, and symptoms include increased expression of inflammatory cytokines, weight loss, decreased colon length, abdominal pain, fever, diarrhea, and/or bleeding. It may be accompanied by repeated worsening and improvement of symptoms, but is not limited to this. Specifically, the inflammatory bowel disease may be at least one selected from the group consisting of ulcerative colitis, Crohn's disease, and Bechet's disease, but is not limited thereto.

When administered to an individual, the combination or composition according to the present invention exhibits one or more of inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration, thereby preventing, improving, or treating enteric diseases. However, it is not limited to this.

When administered to an individual, the combination or composition according to the present invention can prevent, improve, or treat intestinal diseases by causing at least one of an increase in the length of the small intestine, a decrease in inflammation in the small intestine, an increase in the length of the large intestine, and a decrease in inflammation in the large intestine. However, it is not limited to this.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent. These pharmaceutically acceptable carriers, excipients, or diluents may be non-naturally occurring.

In the present invention, the term "pharmaceutically acceptable" means a sufficient amount to produce a therapeutic effect and not to cause side effects, and refers to the type of disease, the patient's age, weight, health, gender, and the patient's sensitivity to the drug. , can be easily determined by a person skilled in the art according to factors well known in the medical field, such as administration route, administration method, administration frequency, treatment period, combination, or drugs used simultaneously. The pharmaceutical composition of the present invention may further include pharmaceutically acceptable excipients. The excipients are not particularly limited thereto, but for oral administration, binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. may be used. For injections, buffers, preservatives, Analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.

The formulation of the composition of the present invention can be prepared in various ways by mixing it with pharmaceutically acceptable excipients as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, preservatives, etc. may be additionally included.

In addition, the pharmaceutical composition of the present invention is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried preparations, and suppositories. It may have a formulation of .

In addition, the composition is formulated into a unit dosage form suitable for administration into the patient's body according to a method commonly used in the pharmaceutical field, specifically a formulation useful for the administration of protein drugs, and is administered as commonly used in the art. Oral, intradermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, digestive tract, topical, sublingual, vaginal, or It may be administered by parenteral administration routes including the rectal route, but is not limited thereto.

In addition, the conjugate can be used by mixing with various pharmaceutically acceptable carriers, such as physiological saline or organic solvents, and to increase stability or absorption, carbohydrates such as glucose, sucrose or dextran, and ascorbic acid. Antioxidants such as acid or glutathione, chelating agents, low-molecular-weight proteins, or other stabilizers can be used as drugs.

The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined depending on the type of drug that is the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender and weight, and the severity of the disease. Specifically, the composition of the present invention may include the insulin secreting peptide, GLP-2, and/or TNFα inhibitor in a pharmaceutically effective amount, but is not limited thereto.

Including the insulin secreting peptide, GLP-2, and/or TNFα inhibitor in a pharmaceutically effective amount means the degree to which the desired pharmacological activity due to the insulin secreting peptide, GLP-2, and/or TNFα inhibitor can be obtained, , It may also mean a pharmaceutically acceptable level at which toxicity or side effects do not occur or are minimal in the administered subject, but is not limited thereto. Such a pharmaceutically effective amount can be determined by comprehensively considering the number of administrations, patient, dosage form, etc.

Although not specifically limited thereto, the pharmaceutical composition of the present invention may contain the ingredients (active ingredients) in an amount of 0.01 to 99% by weight by volume.

The total effective amount of the composition of the present invention can be administered to a patient in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Specifically, the preferred total dose of the peptide or conjugate of the present invention may be about 0.0001 mg to 500 mg per kg of patient body weight per day, but is not limited thereto. However, the dose of the conjugate is determined by considering various factors such as the route of administration and number of treatments of the pharmaceutical composition, as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate. Therefore, taking this into account, anyone skilled in the art will be able to determine an appropriate effective dosage according to the specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

The pharmaceutical composition of the present invention has excellent in vivo persistence and potency, and can significantly reduce the number and frequency of administration of the pharmaceutical agent of the present invention, but is not limited thereto.

One embodiment of the present invention is a food composition for preventing or improving intestinal diseases containing GLP-2, wherein the food composition is administered in combination with an insulin secreting peptide, a TNFα inhibitor, or both. A food composition is provided.

The insulin secreting peptide, GLP-2, TNFα inhibitor, prevention, improvement, and intestinal disease are as described above.

The food composition can be used as a health functional food. When using the composition of the present invention as a food supplement, insulin secreting peptide, GLP-2, long-acting conjugate thereof, TNFα inhibitor, or a combination thereof can be added as is or used with other foods or food ingredients, and can be used by conventional methods. It can be used appropriately. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

The term "health functional food" in the present invention refers to a food manufactured or processed using specific ingredients as raw materials or by extracting, concentrating, refining, or mixing specific ingredients contained in food raw materials for the purpose of health supplementation. It refers to a food designed and processed so that the above ingredients can sufficiently exert bioregulatory functions such as biodefense, regulation of biorhythm, prevention and recovery of disease, etc. to the living body. The composition for health food is used to prevent and treat diseases. It can perform functions related to recovery, etc.

Another aspect embodying the present invention provides a method for preventing, improving, or treating intestinal disease, comprising administering GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both to an individual in need thereof. do.

In another embodiment, it may be a method of preventing, improving, or treating intestinal disease comprising administering and/or using the combination, pharmaceutical composition, or pharmaceutical kit of the present invention to an individual in need thereof. .

In another embodiment, a composition containing a pharmaceutically effective amount of GLP-2, a composition containing a pharmaceutically effective amount of an insulinotropic peptide, a composition containing a pharmaceutically effective amount of a TNFα inhibitor, or both are required. It is a method for preventing, improving, or treating intestinal disease, comprising the steps of combined administration and/or combined use to an individual.

The insulin secreting peptide, GLP-2, TNFα inhibitor, combination, prevention, treatment, improvement, intestinal disease, combination, pharmaceutical composition and pharmaceutical kit are as described above.

In the present invention, the subject is a subject suspected of having an intestinal disease, and the subject suspected of having an enteric disease refers to a mammal, including rats, livestock, etc., including humans that have or may develop the disease, but the GLP- Subjects that can be treated with a combination of 2 and insulin secreting peptide, TNFα inhibitor, or both are included without limitation. Additionally, by administering GLP-2, an insulin secreting peptide, a TNFα inhibitor, or both of the present invention to a subject suspected of having an intestinal disease, the subject can be treated efficiently. Intestinal diseases are the same as described above.

The method of the present invention may include administering a pharmaceutically effective amount of a combination or pharmaceutical composition comprising GLP-2, an insulinotropic peptide, a TNFα inhibitor, or both. The method according to the present invention may be, but is limited to, administering GLP-2, insulin secreting peptide, TNFα inhibitor, or both as a single agent, or administering individual agents simultaneously, separately, sequentially, or in reverse order. It doesn't work.

The appropriate total daily usage amount can be determined by the treating physician within the scope of sound medical judgment, and can be administered once or in several divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a specific patient depends on the type and degree of response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, and general health status, It is desirable to apply it differently depending on various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field.

Another aspect embodying the present invention is the use of the combination, pharmaceutical composition, or pharmaceutical kit for preventing, improving, or treating intestinal diseases, and/or manufacturing a medicament for preventing, improving, or treating intestinal diseases. It is for use. The prevention, improvement, treatment, intestinal diseases, combinations, pharmaceutical compositions and pharmaceutical kits are as described above.

Hereinafter, the present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the present invention and the scope of the present invention is not limited thereto.

Example 1: Preparation of GLP-1 derivative long-acting conjugate

1-1. Preparation of CA-Exendin-4-PEG conjugate

CA-Exendin4 (N-[2-(1H-Imidazol-5-yl)acetyl]-Exendin4, Hanmi Fine Chemical, Korea) has an amine group at the 27th lysine residue in the peptide and an aldehyde (ALD) group at both ends. PEGylated peptide conjugate (mono-PEGylated CA-Exendin-4) through covalent bonding between polyethylene glycol (3.4 kDa, ALD(2) PEG, Hanmi Fine Chemical, Korea) was manufactured. Specifically, the molar ratio of CA-Exendin-4:ALD(2) PEG is 1:5 to 1:15, the CA-Exendin-4 concentration is 6 to 12 mg/ml, at 4 to 10°C to room temperature, about The reaction was allowed for 4 to 12 hours. At this time, the reaction was carried out in 0.1 M HEPES pH 7.0 to 8.5 and about 45% isopropanol, and sodium cyanoborohydride (NaCNBH3) at a concentration of 2 to 5 mM was added. The reaction solution was purified using a Source 15S (Cytiva, USA) column using a buffer containing sodium citrate (pH 2.0 ~ 3.5), approximately 45% ethanol, and a potassium chloride concentration gradient.

1-2. Preparation of CA-Exendin-4-PEG-Fc conjugate

In order to prepare the CA-Exendin-4-PEG-Fc conjugate, an immunoglobulin Fc fragment was added to the mono-PEGylated CA-Exendin-4 obtained through Example 1-1 to increase the total protein concentration to 10 to 50 mg/day. After making it into ml, it was reacted at 4 to 10°C to room temperature for about 12 to 17 hours. At this time, the reaction solution was 0.1 M potassium phosphate, pH 5.5 to 8.5, and 2 to 50 mM sodium cyanoborohydride, a reducing agent, was added. After the reaction was completed, the CA-Exendin-4-PEG-Fc conjugate in the reaction solution was purified using two types of hydrophobic column and anion exchange column. In the case of Source 15Phenyl (Cytiva, USA), a hydrophobic binding column, unreacted immunoglobulin Fc fragments were separated and purified using Tris-HCl (pH 7.5) buffer and NaCl concentration gradient, and Source 15Q (Cytiva, USA), an anion exchange column. In the United States), the CA-Exendin-4-PEG-Fc conjugate was separated and purified by removing overreacted impurities using Tris-HCl (pH 7.5) buffer and NaCl concentration gradient.

Example 2: Preparation of GLP-2 derivative long-acting conjugate

2-1. Preparation of CA GLP-2 RK-PEG conjugate

CA GLP-2 RK, a GLP-2 derivative of SEQ ID NO: 4 in Table 1, is modified with ALD(2) PEG, a polyethylene glycol (the hydrogens at both ends are each replaced with a propionaldehyde group (3-oxopropyl group), and the ethylene glycol repeating unit For PEGylation with modified polyethylene glycol with a formula weight of 3.4 kDa (NOF, Japan), the molar ratio of GLP-2 derivative and ALD(2) PEG is 1:5 to 1:20, GLP-2 The derivative concentration was set to 5 to 10 mg/ml and the reaction was performed at 2 to 8°C for 4 to 16 hours. At this time, the reaction was carried out in 20mM HEPES pH 7.5 and ethanol, and 20mM sodium cyanoborohydride, a reducing agent, was added. In the reaction solution, the monopegylated GLP-2 derivative was purified using a buffer solution containing sodium citrate pH 2.0 and ethanol and a Source 15S (GE, USA) column using a potassium chloride concentration gradient.

2-2. Preparation of CA GLP-2 RK-PEG-Fc conjugate

Next, the molar ratio between the purified monopegylated GLP-2 derivative and the immunoglobulin Fc fragment was set to 1:2 to 1:6, and the total protein concentration was set to 30 to 35 mg/mL at 2 to 8°C. The reaction was performed for 12 to 20 hours. At this time, the reaction solution was 100mM potassium phosphate buffer (pH 6.0) and isopropanol, and 20mM sodium cyanoborohydride was added as a reducing agent.

After the reaction is completed, the reaction solution is applied to a Source 15Q (GE, USA) column using a bis-Tris pH 6.5 buffer and a sodium chloride concentration gradient, and a concentration gradient of ammonium sulfate and sodium citrate pH 5.0 to 5.2. Using Source 15 ISO (GE, USA), a long-acting conjugate of a GLP-2 derivative, which is a conjugate of a GLP-2 derivative covalently linked to an immunoglobulin Fc fragment by a polyethylene glycol linker, was purified.

Example 3: Confirmation of M1 polarization inhibition, macrophage differentiation inhibition, and monocyte migration inhibition effects in THP-1 cells (Monocytes) through combined treatment of GLP-1 derivative long-acting conjugate and GLP-2 derivative long-acting conjugate

Inhibition of M1 polarization, inhibition of macrophage differentiation, and monocyte migration in THP-1 cells (monocytes) resulting from combined treatment with the GLP-1 derivative long-acting conjugate and GLP-2 derivative long-acting conjugate, respectively. To confirm the effect of inhibiting migration, in vitro as follows: An experiment was conducted.

[M1 polarization inhibition confirmation experiment]

GLP-1 derivative long-acting conjugate (0.5 or 1 μM), GLP-2 derivative long-acting conjugate (10 μM), and GLP-1 derivative long-acting conjugate (0.5 μM) + GLP-2 derivative long-acting conjugate to THP-1 cells. Conjugate (10 μM) was treated for 4 hours. In order to induce inflammation, 1 μg/mL of lipopolysaccharides (LPS) was treated for 2 hours, RNA was extracted, and TNF-, a pro-inflammatory cytokine (M1 polarized), was extracted. The mRNA expression levels of α, IL-1β, and IL-6 were measured through qPCR.

[Experiment to confirm inhibition of macrophage differentiation]

GLP-1 derivative long-acting conjugate (0.1 or 1 μM), GLP-2 derivative long-acting conjugate (10 μM), and GLP-1 derivative long-acting conjugate (0.1 μM) + GLP-2 derivative long-acting conjugate to THP-1 cells. The conjugate (10 μM) was treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce THP-1 differentiation. And differentiated cells, adherent cells, were counted.

[Monocyte migration inhibition experiment]

GLP-1 derivative long-acting conjugate (0.1 or 1 μM), GLP-2 derivative long-acting conjugate (10 μM), and GLP-1 derivative long-acting conjugate (0.1 μM) + GLP-2 derivative long-acting conjugate to THP-1 cells. Conjugate (10 μM) was treated for 48 hours. After transferring the drug-treated THP-1 cells to the upper chamber of the Boyden chamber, 50 ng/mL of CCL-2, which can induce movement to the bottom chamber, was added 4 It was cultured for some time. And the degree of migration was measured using a migration assay kit (Abcam).

Through the above experiment, M1 polarization (Figure 1, A), macrophage differentiation (Figure 1, B), and inhibition of monocyte migration (Figure 1, C) of the GLP-1 derivative persistent conjugate and GLP-2 derivative persistent conjugate, respectively. ) The effect was confirmed, and it was confirmed that this effect was further amplified when administered in combination.

Example 4: Confirmation of increased small intestine length and reduced small intestine inflammation in rats with inflammatory bowel disease caused by indomethacin following combined administration of GLP-1 derivative long-acting conjugate, GLP-2 derivative long-acting conjugate, and Anti-TNFα mAb

The effect of improving small intestine length and inflammation in vivo following combined administration of GLP-1 derivative long-acting conjugate, GLP-2 derivative long-acting conjugate, and Anti-TNFα mAb (BioXcell, Catalog # BE0244) For measurement, rats with inflammatory bowel disease caused by indomethacin (INN) were used. Specifically, 8-week-old male rats (SD rats) were divided into 6 groups per group as follows:

- G1: Normal control group (group without drug administration, Vehicle)

- G2: Indomethacin control group (Indomethacin 7.5 mg/kg; Day 1 and Day 2 (D1,2))

- G3: Group administered indomethacin after administration of GLP-2 derivative long-acting conjugate (3.1 mg/kg/single) (GLP-2 derivative long-acting conjugate 3.1 mg/kg/single; Day 0 (D0) + Indomethacin 7.5 mg/kg; D1,2)

- G4: A group administered indomethacin after administration of a GLP-1 derivative long-acting conjugate (0.178 mg/kg/single) (GLP-1 derivative long-acting conjugate 0.178 mg/kg/single; D0 + Indomethacin 7.5 mg/kg; D1,2)

- G5: Group administered indomethacin after administration of GLP-2 derivative long-acting conjugate (3.1 mg/kg/single) and GLP-1 derivative long-acting conjugate (0.178 mg/kg/single) ([GLP-2 derivative long-acting conjugate) Type conjugate 3.1 mg/kg/single + GLP-1 derivative long-acting conjugate 0.178 mg/kg/single; D0] + Indomethacin 7.5 mg/kg, D1,2)

- G6: Group administered Anti-TNFα mAb (0.2 mg/kg/single) followed by indomethacin (Anti-TNFα mAb 0.2 mg/kg/single; D0 + Indomethacin 7.5 mg/kg; D1,2)

- G7: Group administered indomethacin after administration of GLP-2 derivative long-acting conjugate (3.1 mg/kg/single) and Anti-TNFαmAb (0.2 mg/kg/single) ([GLP-2 derivative long-acting conjugate 3.1 mg /kg/single + Anti-TNFα mAb 0.2 mg/kg/single; D0] + Indomethacin 7.5 mg/kg; D1,2)

- G8: Delivery after administration of GLP-2 derivative long-acting conjugate (3.1 mg/kg/single), GLP-1 derivative long-acting conjugate (0.178 mg/kg/single) and Anti-TNFαmAb (0.2 mg/kg/single) Group administered methacin ([GLP-2 derivative long-acting conjugate 3.1 mg/kg/single + GLP-1 derivative long-acting conjugate 0.178 mg/kg/single + Anti-TNFαmAb 0.2 mg/kg/single; D0] + Indomethacin 7.5 mg/kg; D1,2)

In this specification, the value expressed as the dosage of the GLP-1 derivative long-acting conjugate or GLP-2 derivative long-acting conjugate refers to the polyethylene glycol linker portion in the total mass of the GLP-1 derivative long-acting conjugate or GLP-2 derivative long-acting conjugate used. It is a value excluding the mass of , that is, a value expressed based on the sum of the masses of only the polypeptide parts.

And on the third day, the length of the small intestine and the inflammation area (ulcer area) were measured through Evans blue staining through autopsy.

Compared to the indomethacin control group in G2, in the group administered indomethacin after combined administration of GLP-2 derivative long-acting conjugate and GLP-1 derivative long-acting conjugate in G5, the length of the small intestine increased by +25 cm (Figure 2) , the inflammation area (ulcer area) decreased by 6.7% (Figure 3). Compared to the indomethacin control group in G2, in the group administered indomethacin after combined administration of GLP-2 derivative long-acting conjugate and Anti-TNFα mAb in G7, the length of the small intestine increased by +12 cm (Figure 2), and the area of inflammation increased by +12 cm. decreased by 3.5% (Figure 3). In addition, compared to the indomethacin control group in G2, in the group administered indomethacin after combined administration of GLP-2 derivative long-acting conjugate, GLP-1 derivative long-acting conjugate, and Anti-TNFα mAb in G8, the small intestine length was + It increased by 32 cm (similar to the normal control group, Figure 2), and the area of inflammation decreased by 7.2% (similar level to the normal control group, Figure 3).

Example 5: Confirmation of increase in colon length and decrease in ulcerative colitis activity index in mice with ulcerative colitis caused by dextran sulfate sodium following concurrent administration of long-acting GLP-1 derivative and long-acting GLP-2 derivative conjugate

To measure the effect of improving the in vivo large intestine length and ulcerative colitis activity index (DAI) by combined administration of a GLP-1 derivative long-acting conjugate and a GLP-2 derivative long-acting conjugate, dextran Mice with ulcerative colitis caused by sodium sulfate (Dextran sulfate sodium, DSS) were used. Specifically, 7-week-old male mice (C57BL/6) were divided into 10 groups per group as follows. DSS was administered by mixing 2% in drinking water, and one cycle means drinking water containing DSS for 5 days and regular drinking water for 5 days. The drug was administered after a total of 3 cycles, and the first day on which drinking water containing DSS was administered in the first cycle was designated as day 0.

- G1: Normal control group (group without drug administration, Vehicle)

- G2: DSS control group (2% DSS, 3 cycles)

- G3: Group administered DSS followed by GLP-1 derivative long-acting conjugate (0.301 mg/kg/Q2D) (2% DSS, 3 cycles + GLP-1 derivative long-acting conjugate 0.301 mg/kg/Q2D, 2 weeks)

- G4: Group administered DSS followed by GLP-2 derivative long-acting conjugate (1.757 mg/kg/Q2D) (2% DSS, 3 cycles + GLP-2 derivative long-acting conjugate 1.757 mg/kg/Q2D, 2 weeks)

- G5: Group administered GLP-1 derivative long-acting conjugate (0.301 mg/kg/Q2D) and GLP-2 derivative long-acting conjugate (1.757 mg/kg/Q2D) after DSS administration (2% DSS, 3 cycles + [ GLP-1 derivative long-acting conjugate 0.301 mg/kg/Q2D + GLP-2 derivative long-acting conjugate 1.757 mg/kg/Q2D], 2 weeks)

- G6: Group administered DSS followed by Cyclosporine A (20 mg/kg/QD, PO) (2% DSS, 3 cycles + Cyclosporine A 20 mg/kg/QD, 2 weeks)

And on day 44, improvement in the condition was measured by measuring the length of the large intestine through autopsy (Figure 4).

Ulcerative colitis activity index was measured by scoring stool consistency and bleeding status. In case of stool consistency, it is normal (0 points), the shape is visible to the naked eye, but when pressed, it rubs gently unlike the normal group stools (1 point), and unlike the normal group stools, the shape is not round and is gently rubbed (2 points), shape. It was measured as not being held together and the stools were very soft (3 points), and the area around the anus was wet due to diarrhea and the stools did not come out easily and water came out (4 points). In the case of bleeding conditions, there is no bleeding (0 points), blood is visible when the stool is pressed (2 points), blood is visible without the need to push the stool (3 points), and severe bleeding is confirmed from the rectum. It was measured by condition (4 points). Measurements were conducted on days 21, 23, 25, 28, 30, 32, 35, 37, 39, 42, and 44, and improvement in symptoms was measured by adding up the values ( Figure 5).

Compared to the DSS control group in G2, the length of the colon increased by about +0.8cm (18%) in the group administered GLP-1 derivative long-acting conjugate and GLP-2 derivative long-acting conjugate after DSS administration in G5 (compared to the normal control group). At a similar level, Figure 4), the ulcerative colitis activity index decreased by 10 (Figure 5). In addition, compared to the group administered Cyclosporine A (20 mg/kg/QD), which is used in clinical practice immediately before surgery (severe) after DSS administration in G6, after DSS administration in G5, GLP-1 derivative long-acting conjugate and GLP- 2 In the group administered the derivative long-acting conjugate, the length of the colon increased by approximately +1.4 cm (25%) (similar to the normal control group, Figure 4), and the ulcerative colitis activity index decreased by 6 (Figure 5).

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (28)

A pharmaceutical composition for the prevention or treatment of intestinal diseases containing a pharmaceutically effective amount of GLP-2,
The pharmaceutical composition is characterized in that it is administered in combination with an insulin secreting peptide, a TNFα inhibitor, or both.
The method of claim 1, wherein the insulin secreting peptide is glucagon-like peptide-1 (GLP-1), exendin-3, exendin-4, agonists, derivatives, fragments, variants thereof, and A pharmaceutical composition selected from the group consisting of combinations thereof.
The method of claim 1, wherein the N-terminal histidine residue of the insulin secreting peptide is imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β- A pharmaceutical composition, which is an insulin secreting peptide derivative substituted with carboxyimidazopropionyldeshistidine.
The method of claim 1, wherein the insulin secreting peptide is native exendin-4, exendin in which the N-terminal amino group bound to the alpha carbon and alpha carbon of the histidine residue, which is the first amino acid at the N-terminal of exendin-4, has been removed. -4 derivative, exendin-4 derivative in which the N-terminal amino group of exendin-4 has been removed, exendin-4 derivative in which the N-terminal amino group of exendin-4 has been replaced with a hydroxyl group, N of exendin-4 -Exendin-4 derivatives in which the terminal amino group is modified with two methyl groups, Exendin-4 derivatives in which the N-terminal amino group of Exendin-4 is replaced with a carboxyl group, and the twelfth amino acid (lysine) of Exendin-4 is changed to serine. A pharmaceutical composition comprising a substituted exendin-4 derivative, or an exendin-4 derivative in which the twelfth amino acid (lysine) of exendin-4 is substituted with arginine.
The pharmaceutical composition according to claim 1, wherein the GLP-2 is native GLP-2 or a GLP-2 derivative.
The method of claim 5, wherein the GLP-2 derivative is a group consisting of substitution, addition, deletion, modification, and combinations thereof for at least one amino acid in the native GLP-2 sequence. A pharmaceutical composition that is a GLP-2 derivative with a modification selected from .
The pharmaceutical composition according to claim 5, wherein the GLP-2 derivative has a modification in at least one amino acid among amino acids 1, 2, 30, and 33 in SEQ ID NO: 1.
The pharmaceutical composition according to claim 5, wherein the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 1:
[General Formula 1]
_{X 1 _ _}
here,
X ₁ is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine;
X ₂ is alanine, glycine, or Aib(2-aminoisobutyric acid);
X ₃₀ is lysine or arginine;
X ₃₄ is absent or is lysine, arginine, glutamine, histidine, 6-azidolysine or cysteine;
However, among the amino acid sequences of General Formula 1, sequences identical to SEQ ID NO: 1 are excluded.
The method of claim 8, wherein the GLP-2 derivative is
(1) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is cysteine,
(2) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is lysine,
(3) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is arginine, and X ₃₄ is lysine,
(4) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is lysine, and X ₃₄ is 6-azidolysine, or
(5) X ₁ is imidazoacetyldeshistidine, X ₂ is glycine, X ₃₀ is arginine, and X ₃₄ is cysteine,
(6) X ₁ is imidazoacetyldeshistidine, X ₂ is Aib, X ₃₀ is lysine, and X ₃₄ is cysteine, or
(7) A pharmaceutical composition wherein X ₁ is histidine, X ₂ is Aib, X ₃₀ is lysine, and X ₃₄ is cysteine.
The pharmaceutical composition according to claim 5, wherein the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 2:
[General Formula 2]
_{X 1 _ _}
here,
X ₁ is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine;
X ₂ is alanine, glycine, or Aib(2-aminoisobutyric acid);
X ₃₀ is lysine or arginine;
X ₃₄ is one or more optional amino acids or one or more optional amino acids upon which a modification has occurred;
However, among the amino acid sequences of General Formula 2, sequences identical to SEQ ID NO: 1 are excluded.
The pharmaceutical composition according to claim 5, wherein the GLP-2 derivative is a peptide with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8.
The pharmaceutical composition according to claim 1, wherein GLP-2 and insulin secreting peptide are administered in combination.
The pharmaceutical composition according to claim 1, wherein GLP-2 and TNFα inhibitor are administered in combination.
The pharmaceutical composition according to claim 1, wherein GLP-2, insulin secreting peptide, and TNFα inhibitor are administered in combination.
The pharmaceutical composition of claim 1, wherein the TNFα inhibitor is a soluble TNF receptor, an anti-TNFα antibody or fragment thereof, or a combination thereof.
The pharmaceutical composition of claim 1, wherein the intestinal disease is at least one selected from the group consisting of irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, and intestinal damage.
The pharmaceutical composition according to claim 16, wherein the inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, and Behcet's disease.
The pharmaceutical composition of claim 1, wherein the composition exhibits one or more of inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration when administered to a subject.
The pharmaceutical composition of claim 1, wherein, when administered to a subject, the composition causes at least one of an increase in the length of the small intestine, a decrease in inflammation in the small intestine, an increase in the length of the large intestine, and a decrease in inflammation in the large intestine.
The pharmaceutical composition according to claim 1, wherein the insulin secreting peptide and GLP-2 are unmodified or amidated at their C-terminus.
According to paragraph 1,
(i) the insulin secreting peptide is in the form of a long-acting conjugate in which a biocompatible material capable of increasing its in vivo half-life is bound, or
(ii) The GLP-2 is in the form of a long-acting conjugate combined with a biocompatible material that can increase its in vivo half-life, or
(iii) A pharmaceutical composition in the form of a long-acting conjugate in which the insulin secreting peptide and GLP-2 are each combined with a biocompatible material capable of increasing their in vivo half-life.
The pharmaceutical composition according to claim 21, wherein the conjugate is represented by the following formula (1):
[Formula 1]
X - La - F
here,
X is insulinotropic peptide or GLP-2;
L is a linker containing ethylene glycol repeating units;
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
F is the immunoglobulin Fc region;
The “-” above is a covalent bond.
The pharmaceutical composition according to claim 22, wherein the immunoglobulin Fc region is a non-glycosylated IgG4 Fc region.
The pharmaceutical composition according to claim 22, wherein F is a dimer composed of two polypeptide chains, and one end of L is linked to only one polypeptide chain of the two polypeptide chains.
The pharmaceutical composition of claim 22, wherein L is polyethylene glycol.
The pharmaceutical composition according to claim 22, wherein the formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.
The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
28. The method of any one of claims 1 to 27, comprising: (i) GLP-2 and insulinotropic peptide; (ii) GLP-2 and TNFα inhibitors; or (iii) a pharmaceutical composition characterized in that GLP-2, an insulinotropic peptide, and a TNFα inhibitor are administered in combination simultaneously, sequentially, or in reverse order.